Enzymes which use polynucleotides as substrates are often fundamental to the biochemical function of many organisms and viruses. This group of enzymes includes, for example, endonucleases, exonucleases and ribozymes. A number of enzymes act on substrates to give single-stranded oligonucleotide products. For example, the influenza virus endonuclease cleaves capped host cell transcripts 10 to 13 bases from the 5' end. Sequence specific RNA endonucleases are known; for example the 2-5A-dependent RNase found in higher animals functions to cleave single-stranded regions of RNA 3' of UpNp dimers, with a preference for UU and UA sequences. In addition, ribozymes are known to cleave single-stranded RNAs at specific recognition sequences.
While it is recognized that inhibitors or activators of these enzymes might be new classes of therapeutic or preventative compounds, particularly against viral diseases, identification of such compounds has been hampered by the lack of a convenient assay system.
An ideal enzyme assay system should have: a) high throughput; b) the ability to distinguish enzyme-catalyzed cleavage from nonspecific nucleotide cleavage; and c) high sensitivity. Previous nucleotide cleavage assays involved the use of polyacrylamide gel electrophoresis to separate product from substrate (Plotch et al., 1981 Cell 23:847-858) which is not convenient for processing large numbers of samples.